1. Field of the Invention
The present invention provides and includes monoclonal antibodies (MoAbs or mAbs) specific for ALPHA-ACTININ-4 antigens, hybridoma lines that secrete these ALPHA-ACTININ-4 mAbs or fragments thereof, and the use of such mAbs or fragments thereof to detect ALPHA-ACTININ-4 antigens, particularly those expressed by cancer cells. The present invention also includes chimeric and humanized antibodies based upon these mAbs, processes for producing monoclonal, chimeric, and humanized antibodies using recombinant DNA technology, and their therapeutic uses, particularly in the treatment of cancer.
2. Description of the Background Art
Antibodies (also referred to as immunoglobulins) are constructed from four polypeptide chains, two heavy chains and two light chains. The two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (γ) and kappa (κ). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.
Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors.
The variable domain is responsible for antigen-specific binding, and the constant domains carry out effector functions. The variable domain is divided into complementarity determining regions (CDR1, CDR2 and CDR3) and framework regions (FWR1, FWR2, and FWR3). Using the Kabat residue numbering system, CDRs 1, 2, and 3 are delineated by amino acid positions 31-35, 50-65, and 95-102 for heavy chains, and amino acid positions 24-34, 50-56, and 89-97 for light chains. While these amino acid positions define the boundaries of each CDR, the lengths of the CDRs can vary. The CDRs create the antigen binding pocket of the molecule through the interaction between heavy and light chain variable regions while the framework regions provide the scaffolding on which the antigen binding pocket sits. Occasionally, residues from nonhypervariable or framework regions (FWRs) influence the overall domain structure and hence the combining site.
There are two major methods for generating vertebrate antibodies: generation of polyclonal antibodies in situ by mammalian B lymphocytes and generation of monoclonal antibodies in cell culture by B cell hybrids. To generate antibodies in situ, an animal (such as a mouse or rabbit) is injected with an antigen. Several weeks later, blood is drawn from the animal and centrifuged. The resulting serum contains antibodies against the injected antigen. The resulting antibodies are polyclonal antibodies because they are products of many different populations of antibody producing cells and hence differ somewhat in their precise specificity and affinity for the antigen.
Monoclonal antibodies are produced using hybridoma technology in which an antibody producing cell is fused with a tumor cell that has the capacity for unlimited proliferation. In contrast to polyclonal antibodies, monoclonal antibodies are homogeneous because they are synthesized by a population of identical cells that are derived from a single hybridoma cell.
However, the use of monoclonal antibodies in humans is severely restricted when the monoclonal antibody is produced in a non-human animal. Repeated injections in humans of a “foreign” antibody, such as a mouse antibody, may lead to harmful hypersensitivity reactions, i.e., anti-mouse antibody (HAMA) or an anti-idiotypic, response. The HAMA response makes repeated administrations ineffective due to an increased rate of clearance from the patient's serum and/or allergic reactions by the patient.
Attempts have been made to manufacture human-derived monoclonal antibodies using human hybridomas. Unfortunately, yields of monoclonal antibodies from human hybridoma cell lines are relatively low compared to mouse hybridomas. Additionally, human cell lines expressing immunoglobulins are relatively unstable compared to mouse cell lines, and the antibody producing capability of these human cell lines is transient. Thus, while human immunoglobulins are highly desirable, human hybridoma techniques have not yet reached the stage where human monoclonal antibodies with the required antigenic specificities can be easily obtained.
Thus, antibodies of non-human origin are typically genetically engineered to create chimeric or humanized antibodies. Such genetic engineering results in antibodies with a reduced risk of a HAMA response compared to that expected after injecting a human patient with a mouse antibody. For example, chimeric antibodies can be formed by grafting non-human variable regions to human constant regions. Humanized antibodies are formed by grafting non-human complementarity determining regions (CDRs) onto human framework regions (FWRs). Typically, humanized monoclonal antibodies are formed by grafting all six (three light chain and three heavy chain) CDRs from a non-human antibody into FWRs of a human antibody. However, these modified antibodies still retain various non-human light and heavy chain variable regions: the chimeric antibodies retain entire non-human variable regions, and CDR-grafted antibodies retain CDR of non-human origin. Such non-human regions can elicit an immunogenic reaction when administered to a human patient. Thus, many humanized mAbs remain immunogenic.
It has been shown that not all residues of CDRs are critical in the complementarity of antigen/antibody surfaces. Known structures of the antigen-antibody complexes suggest that only 20-33% of CDR residues are involved in antigen contact. A comprehensive analysis of the available data of the sequences and the three dimensional structure of antibody combining sites can be used to identify CDR residues that may be critical in the antigen antibody interaction. These residues are designated as specificity determining residues (SDRs), and they may be shared among antibodies to a particular antigen.
During the process of oncogenesis, a number of cell-surface molecules or markers appear on cells. Such tumor-related markers may include, but are not limited to, oncofetoproteins, neoglycoproteins, sphignolipids, and modifications of existing surface proteins. Such new or altered structures are often shed from the tumor cell surface and appear in the serum or in other biological fluids. The detection of any of these substances or “tumor markers” or “biomarkers” serves as the basis for diagnosing or monitoring the progress of neoplastic disease.
Using monoclonal antibody (mAb or MoAb) technology, it has become possible to obtain pure antibody populations which permit better purification and characterization of the various tumor markers and tumor-associated antigens that are useful for immunodiagnosis or immunotherapy. Many mAbs have been described that have varying degrees of selectivity for tumor antigens (versus normal cell surface markers); some of these tumor antigens are broadly represented across several or many tumor types, whereas others appear to be truly tumor or cancer cell-specific.
ALPHA-ACTININ-4 may be a useful marker for the detection of neuroendocrine pulmonary tumors (NEPT) (see N. H. Cho et al., J. Proteome Res. 5(3):643-50 (2006)), and for the detection of hepatocarcinoma (HCC) metastasis (see Z. Dai et al., Proteomics 6(21):5857-67 (2006)).
Accordingly, there is a need for an antibody molecule to selectively detect diseases, such as solid tumors, characterized by the expression or localization of ALPHA-ACTININ-4 gene products that can be used repeatedly and produced easily and efficiently. There is also a need for an antibody molecule which has high affinity for gene products of ALPHA-ACTININ-4 and homologues thereof.